In situ cryo-electron tomography reveals local cellular machineries for axon branch development

Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon branching allows to form new paths and increases circuit complexity. However, our understanding of branch formation is sparse due to technical limitations. Using in situ cellular cryo-electron tomography on primary mouse neurons, we directly visualized the remodeling of organelles and cytoskeleton structures at axon branches. Strikingly, branched areas functioned as hotspots concentrating organelles to support dynamic activities. Unaligned actin filaments assembled at the base of premature branches and remained while filopodia diminished. Microtubules and ER co-migrated into preformed branches to support outgrowth together with accumulating compact ~500 nm mitochondria and locally clustered ribosomes. We obtained a roadmap of events and present the first direct evidence of local protein synthesis selectively taking place at axon branches, allowing to serve as unique control hubs for axon development and downstream neural network formation.